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The EMBO Journal Vol.17 No.19 pp.5577–5587, 1998Functional dissection ofArabidopsisCOP1 revealsspecific roles of its three structural modules in lightcontrol of seedling developmentKeiko U.Torii1, Timothy W.McNellis2andXing-Wang Deng3Department of Molecular, Cellular and Developmental Biology, YaleUniversity, PO Box 208104, 165 Prospect Street, OML 301, NewHaven, CT 06520-8104, USA1Present address: Department of Biology, 4129 Natural Science Bldg,University of Michigan, Ann Arbor, MI 48109-1048, USA2Present address: Department of Plant Biology, 111 Koshland Hall,University of California at Berkeley, Berkeley, CA 94720, USA3Corresponding authore-mail: [email protected] COP1 acts as a repressor of photomorpho-genesis in darkness, and light stimuli abrogate therepressive ability and nuclear abundance of COP1.COP1 has three known structural modules: an N-terminal RING-finger, followed by a predicted coiled-coil and C-terminal WD-40 repeats. A systematic studywas undertaken to dissect the functional roles of thesethree COP1 domains in light control of Arabidopsisseedling development. Our data suggest that COP1acts primarily as a homodimer, and probably dimerizesthrough the coiled-coil domain. The RING-finger andthe coiled-coil domains can function independently aslight-responsive modules mediating the light-controllednucleocytoplasmic partitioning of COP1. The C-ter-minal WD-40 domain functions as an autonomousrepressor module since the overexpression of COP1mutant proteins with intact WD-40 repeats are able tosuppress photomorphogenic development. This WD-40 domain-mediated repression can be at least in partaccounted for by COP1’s direct interaction with andnegative regulation of HY5, a bZIP transcription factorthat positively regulates photomorphogenesis. How-ever, COP1 self-association is a prerequisite for theobserved interaction of the COP1 WD-40 repeats withHY5. This work thus provides a structural basis ofCOP1 as a molecular switch.Keywords: Arabidopsis/coiled-coil/photomorphogenesis/RING-finger/WD-40 repeatsIntroductionArabidopsis seedlings display contrasting developmentalpatterns in the presence and absence of light. Undernormal light conditions, seedlings follow photomorpho-genic development characterized by inhibition of hypo-cotyl elongation, development of expanded cotyledons,biogenesis of chloroplasts and expression of light-inducible genes. In darkness, seedlings etiolate, displayingelongated hypocotyls, closed and unexpanded cotyledons,and apical hooks. Also, the light-inducible genes arerepressed, and plastids develop into non-photosynthetic© Oxford University Press5577etioplasts in the darkness. This developmental commitmentis plastic and reversible; the etiolated seedlings can responddynamically to incoming light stimuli and initiate photo-morphogenesis (for review see Chory, 1993; McNellis andDeng, 1995).It is not fully understood how light stimuli perceivedby multiple photoreceptors are transduced and integratedto affect developmental programs. Genetic screens ofArabidopsis seedlings, based on either etiolated pheno-types under light conditions or photomorphogenic pheno-types in complete darkness, have identified a large numberof the light-signal transduction components involved incontrolling seedling development (for review see McNellisand Deng, 1995). Mutant seedlings with reduced light-responsiveness display characteristic long hypocotyl (hy)phenotypes. This class of mutants defines positive regu-lators of photomorphogenesis including photoreceptors(e.g. phyA, phyB and hy4) and components acting down-stream of specific photoreceptor (e.g. fhy1, fhy3 andred1) or multiple photoreceptors (e.g. hy5). (Chory, 1992;Whitelam et al., 1993; Wagner et al., 1997). The recentmolecular identification of HY5 as a bZIP transcriptionfactor may provide a tool to bridge the light-signaltransduction pathway to the control of gene expression(Oyama et al., 1997). The second class of mutants includesthose that display constitutive photomorphogenesis,namely constitutive photomorphogenic (cop), de-etiolated(det) and fusca (fus) mutants (reviewed by Wei and Deng,1996). Genetic studies indicate that their gene productsfunction as negative regulators acting downstream ofmultiple photoreceptors, including phyA, phyB and theblue-light receptor CRY1 (Ang and Deng, 1994; McNellisand Deng, 1995). While a subset of these mutants areimplicated in playing a role in phytohormone signaling(Chory and Li, 1997; Kraepiel and Miginiac, 1997), 10of the pleiotropic and essential COP/DET/FUS loci arebelieved to be responsible for mediating the suppressionof photomorphogenic seedling development in darkness(Wei and Deng, 1996).The molecular identification of four COP/DET/FUSgenes, namely COP1, COP9, DET1 and FUS6 (COP11)provides an opportunity to understand the molecularmechanisms of repression of photomorphogenesis (Denget al., 1992; Castle and Meinke, 1994; Pepper et al., 1994;Wei et al., 1994). COP9, DET1 and FUS6 encode novelα-helical-rich proteins that constitutively localize in thenucleus (Pepper et al., 1994; Wei et al., 1994; Chamovitzet al., 1996; Staub et al., 1996). COP9 has been found tobe a part of an eight-subunit protein complex consistingof COP9, FUS6 (COP11), presumably COP8 and others(Wei et al., 1994, 1998; Chamovitz et al., 1996; Wei andDeng, 1996, 1998). COP1, on the other hand, appears tofunction as an autonomous repressor of photomorpho-genesis based on previous experiments in modulatingK.U.Torii, T.W.McNellis and X-W.DengFig. 1. COP1 dimerizes in vitro and in vivo through the Coil domain. (A) In vitro cross-linking analysis of COP1. The oligomeric nature ofradiolabeled flag-COP1 (indicated by bars at the right side of each panel) was analyzed by using chemical cross-linker EGS and Dsub. Brackets onthe right-hand side of each panel indicate the positions of cross-linked products that correspond to dimers. In-vitro-translated flag-COP1 yielded twobands with close molecular masses, the lower of which may be due to inappropriate translation initiated from an endogenous methionine of COP1.(B) The gel-filtration profiles of the COP1 protein extracted from wild-type seedlings grown under continuous white light or in darkness for 6 days.The proteins in the fractions were separated by SDS–PAGE and examined by protein gel immunoblot. The molecular masses (in kDa) estimated byprotein standards are listed above the appropriate fractions. For experimental details, see


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